Cytolysis by CCR5-using human immunodeficiency virus type 1 envelope glycoproteins is dependent on membrane fusion and can be inhibited by high levels of CD4 expression

T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4(+) CXCR4(+) T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5(+) target cells. As is the case for CXCR4(+) target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4(+) CCR5(+) cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4(+) T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4(+) T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.     

LIM kinase 1 modulates cortical actin and CXCR4 cycling and is activated by HIV-1 to initiate viral infection

Almost all viral pathogens utilize a cytoskeleton for their entry and intracellular transport. In HIV-1 infection, binding of the virus to blood resting CD4 T cells initiates a temporal course of cortical actin polymerization and depolymerization, a process mimicking the chemotactic response initiated from chemokine receptors. The actin depolymerization has been suggested to promote viral intracellular migration through cofilin-mediated actin treadmilling. However, the role of the virus-mediated actin polymerization in HIV infection is unknown, and the signaling molecules involved remain unidentified. Here we describe a pathogenic mechanism for triggering early actin polymerization through HIV-1 envelope-mediated transient activation of the LIM domain kinase (LIMK), a protein that phosphorylates cofilin. We demonstrate that HIV-mediated LIMK activation is through gp120-triggered transient activation of the Rack-PAK-LIMK pathway, and that knockdown of LIMK through siRNA decreases filamentous actin, increases CXCR4 trafficking, and diminishes viral DNA synthesis. These results suggest that HIV-mediated early actin polymerization may directly regulate the CXCR4 receptor during viral entry and is involved in viral DNA synthesis. Furthermore, we also demonstrate that in resting CD4 T cells, actin polymerization can be triggered through transient treatment with a pharmacological agent, okadaic acid, that activates LIMK and promotes HIV latent infection of resting CD4 T cells. Taken together, our results suggest that HIV hijacks LIMK to control the cortical actin dynamics for the initiation of viral infection of CD4 T cells.     

Identification of conserved and variable structures in the human immunodeficiency virus gp120 glycoprotein of importance for CXCR4 binding

CD4 and the chemokine receptors, CXCR4 and CCR5, serve as receptors for human immunodeficiency virus type 1 (HIV-1). Binding of the HIV-1 gp120 envelope glycoprotein to the chemokine receptors normally requires prior interaction with CD4. Mapping the determinants on gp120 for the low-affinity interaction with CXCR4 has been difficult due to the nonspecific binding of this viral glycoprotein to cell surfaces. Here we examine the binding of a panel of gp120 mutants to paramagnetic proteoliposomes displaying CXCR4 on their surfaces. We show that the gp120 beta19 strand and third variable (V3) loop contain residues important for CXCR4 interaction. Basic residues from both elements, as well as a conserved hydrophobic residue at the V3 tip, contribute to CXCR4 binding. Removal of the gp120 V1/V2 variable loops allows the envelope glycoprotein to bind CXCR4 in a CD4-independent manner. These results indicate that although some variable gp120 residues contribute to the specific binding to CCR5 or CXCR4, gp120 elements common to CXCR4- or CCR5-using strains are involved in the interaction with both coreceptors.     

Apoptosis of bystander T cells induced by human immunodeficiency virus type 1 with increased envelope/receptor affinity and coreceptor binding site exposure

Apoptosis of uninfected bystander CD4(+) T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) pathogenesis. The viral and host mechanisms that lead to bystander apoptosis are not well understood. To investigate properties of the viral envelope glycoproteins (Env proteins) that influence the ability of HIV-1 to induce bystander apoptosis, we used molecularly cloned viruses that differ only in specific amino acids in Env. The ability of these strains to induce bystander apoptosis was tested in herpesvirus saimiri-immortalized primary CD4(+) T cells (CD4/HVS), which resemble activated primary T cells. Changes in Env that increase affinity for CD4 or CCR5 or increase coreceptor binding site exposure enhanced the capacity of HIV-1 to induce bystander apoptosis following viral infection or exposure to nonreplicating virions. Apoptosis induced by HIV-1 virions was inhibited by CD4, CXCR4, and CCR5 antibodies or by the CXCR4 inhibitor AMD3100, but not the fusion inhibitor T20. HIV-1 virions with mutant Envs that bind CXCR4 but are defective for CD4 binding or membrane fusion induced apoptosis, whereas CXCR4 binding-defective mutants did not. These results demonstrate that HIV-1 virions induce apoptosis through a CXCR4- or CCR5-dependent pathway that does not require Env/CD4 signaling or membrane fusion and suggest that HIV-1 variants with increased envelope/receptor affinity or coreceptor binding site exposure may promote T-cell depletion in vivo by accelerating bystander cell death.     

Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates.

Primary isolates of human immunodeficiency virus type 1 (HIV-1) are much less sensitive to neutralization by soluble CD4 (sCD4) and sCD4-immunoglobulin (Ig) chimeras (CD4-IgG) than are HIV-1 strains adapted to growth in cell culture. We demonstrated that there are significant reductions (10- to 30-fold) in the binding of sCD4 and CD4-IgG to intact virions of five primary isolates compared with sCD4-sensitive, cell culture-adapted isolates RF and IIIB. However, soluble envelope glycoproteins (gp120) derived from the primary isolate virions, directly by detergent solubilization or indirectly by recombinant DNA technology, differed in affinity from RF and IIIB gp120 by only one- to threefold. The reduced binding of sCD4 to these primary isolate virions must therefore be a consequence of the tertiary or quaternary structure of the envelope glycoproteins in their native, oligomeric form on the viral surface. In addition, the rate and extent of sCD4-induced gp120 shedding from these primary isolates was lower than that from RF. We suggest that reduced sCD4 binding and increased gp120 retention together account for the relative resistance of these primary isolates to neutralization by sCD4 and CD4-IgG and that virions of different HIV-1 isolates vary both in the mechanism of sCD4 binding and in subsequent conformational changes in their envelope glycoproteins.     

HIV-1 envelope is a neutral antagonist to CXCR4 in T-cells

The chemokine receptor CXCR4 is the principal coreceptor for X4 strains of HIV-1. We show that gp120 is unable to induce interactions between CXCR4 and G-protein in T-cells, but antagonized the agonist effect of SDF-1alpha, the natural ligand for CXCR4. Gp120 had ten times lower affinity for CXCR4 than CD4, implying that a substantial role for cellular CD4 may be to facilitate binding of the viral envelope to CXCR4. Binding of gp120 to CXCR4 was neither regulated by guanine nucleotides, nor affected by divalent cations, was temperature independent and bound to a homogenous population of CXCR4, which is characteristic for an antagonist to a G-protein coupled receptor. In contrast, SDF-1alpha binds to two affinity states of CXCR4 in T-cell membranes, which are modulated by guanine nucleotides. Binding of SDF-1alpha to CXCR4 was highly temperature dependent. Thus, the interaction of CXCR4 with HIV-1 viral envelope and chemokine exhibits fundamental differences.     

The HIV-1 Gp120/CXCR4 Axis Promotes CCR7 Ligand-Dependent CD4 T Cell Migration: CCR7 Homo- and CCR7/CXCR4 Hetero-Oligomer Formation as a Possible Mechanism for Up-Regulation of Functional CCR7

During human immunodeficiency virus (HIV) infection, enhanced migration of infected cells to lymph nodes leads to efficient propagation of HIV-1. The selective chemokine receptors, including CXCR4 and CCR7, may play a role in this process, yet the viral factors regulating chemokine-dependent T cell migration remain relatively unclear. The functional cooperation between the CXCR4 ligand chemokine CXCL12 and the CCR7 ligand chemokines CCL19 and CCL21 enhances CCR7-dependent T cell motility in vitro as well as cell trafficking into the lymph nodes in vivo. In this study, we report that a recombinant form of a viral CXCR4 ligand, X4-tropic HIV-1 gp120, enhanced the CD4 T cell response to CCR7 ligands in a manner dependent on CXCR4 and CD4, and that this effect was recapitulated by HIV-1 virions. HIV-1 gp120 significantly enhanced CCR7-dependent CD4 T cell migration from the footpad of mice to the draining lymph nodes in in vivo transfer experiments. We also demonstrated that CXCR4 expression is required for stable CCR7 expression on the CD4 T cell surface, whereas CXCR4 signaling facilitated CCR7 ligand binding to the cell surface and increased the level of CCR7 homo- as well as CXCR4/CCR7 hetero-oligomers without affecting CCR7 expression levels. Our findings indicate that HIV-evoked CXCR4 signaling promotes CCR7-dependent CD4 T cell migration by up-regulating CCR7 function, which is likely to be induced by increased formation of CCR7 homo- and CXCR4/CCR7 hetero-oligomers on the surface of CD4 T cells.     

CD4-Induced Conformational Changes in the Human Immunodeficiency Virus Type 1 gp120 Glycoprotein: Consequences for Virus Entry and Neutralization

Human immunodeficiency virus type 1 (HIV-1) entry into target cells involves sequential binding of the gp120 exterior envelope glycoprotein to CD4 and to specific chemokine receptors. Soluble CD4 (sCD4) is thought to mimic membrane-anchored CD4, and its binding alters the conformation of the HIV-1 envelope glycoproteins. Two cross-competing monoclonal antibodies, 17b and CG10, that recognize CD4-inducible gp120 epitopes and that block gp120-chemokine receptor binding were used to investigate the nature and functional significance of gp120 conformational changes initiated by CD4 binding. Envelope glycoproteins derived from both T-cell line-adapted and primary HIV-1 isolates exhibited increased binding of the 17b antibody in the presence of sCD4. CD4-induced exposure of the 17b epitope on the oligomeric envelope glycoprotein complex occurred over a wide range of temperatures and involved movement of the gp120 V1/V2 variable loops. Amino acid changes that reduced the efficiency of 17b epitope exposure following CD4 binding invariably compromised the ability of the HIV-1 envelope glycoproteins to form syncytia or to support virus entry. Comparison of the CD4 dependence and neutralization efficiencies of the 17b and CG10 antibodies suggested that the epitopes for these antibodies are minimally accessible following attachment of gp120 to cell surface CD4. These results underscore the functional importance of these CD4-induced changes in gp120 conformation and illustrate viral strategies for sequestering chemokine receptor-binding regions from the humoral immune response.     

Migration of antigen-specific T cells away from CXCR4-binding human immunodeficiency virus type 1 gp120

Cell-mediated immunity depends in part on appropriate migration and localization of cytotoxic T lymphocytes (CTL), a process regulated by chemokines and adhesion molecules. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode chemotactically active proteins, suggesting that dysregulation of immune cell trafficking may be a strategy for immune evasion. HIV-1 gp120, a retroviral envelope protein, has been shown to act as a T-cell chemoattractant via binding to the chemokine receptor and HIV-1 coreceptor CXCR4. We have previously shown that T cells move away from the chemokine stromal cell-derived factor 1 (SDF-1) in a concentration-dependent and CXCR4 receptor-mediated manner. Here, we demonstrate that CXCR4-binding HIV-1 X4 gp120 causes the movement of T cells, including HIV-specific CTL, away from high concentrations of the viral protein. This migratory response is CD4 independent and inhibited by anti-CXCR4 antibodies and pertussis toxin. Additionally, the expression of X4 gp120 by target cells reduces CTL efficacy in an in vitro system designed to account for the effect of cell migration on the ability of CTL to kill their target cells. Recombinant X4 gp120 also significantly reduced antigen-specific T-cell infiltration at a site of antigen challenge in vivo. The repellant activity of HIV-1 gp120 on immune cells in vitro and in vivo was shown to be dependent on the V2 and V3 loops of HIV-1 gp120. These data suggest that the active movement of T cells away from CXCR4-binding HIV-1 gp120, which we previously termed fugetaxis, may provide a novel mechanism by which HIV-1 evades challenge by immune effector cells in vivo.     

Molecular Recognition of CXCR4 by a Dual Tropic HIV-1 gp120 V3 Loop

HIV-1 cell entry is initiated by the interaction of the viral envelope glycoprotein gp120 with CD4, and chemokine coreceptors CXCR4 and CCR5. The molecular recognition of CXCR4 or CCR5 by the HIV-1 gp120 is mediated through the V3 loop, a fragment of gp120. The binding of the V3 loop to CXCR4 or CCR5 determines the cell tropism of HIV-1 and constitutes a key step before HIV-1 cell entry. Thus, elucidating the molecular recognition of CXCR4 by the V3 loop is important for understanding HIV-1 viral infectivity and tropism, and for the design of HIV-1 inhibitors. We employed a comprehensive set of computational tools, predominantly based on free energy calculations and molecular-dynamics simulations, to investigate the molecular recognition of CXCR4 by a dual tropic V3 loop. We report what is, to our knowledge, the first HIV-1 gp120 V3 loop:CXCR4 complex structure. The computationally derived structure reveals an abundance of polar and nonpolar intermolecular interactions contributing to the HIV-1 gp120:CXCR4 binding. Our results are in remarkable agreement with previous experimental findings. Therefore, this work sheds light on the functional role of HIV-1 gp120 V3 loop and CXCR4 residues associated with HIV-1 coreceptor activity.     

Molecular Recognition of CXCR4 by a Dual Tropic HIV-1 gp120 V3 Loop

HIV-1 cell entry is initiated by the interaction of the viral envelope glycoprotein gp120 with CD4, and chemokine coreceptors CXCR4 and CCR5. The molecular recognition of CXCR4 or CCR5 by the HIV-1 gp120 is mediated through the V3 loop, a fragment of gp120. The binding of the V3 loop to CXCR4 or CCR5 determines the cell tropism of HIV-1 and constitutes a key step before HIV-1 cell entry. Thus, elucidating the molecular recognition of CXCR4 by the V3 loop is important for understanding HIV-1 viral infectivity and tropism, and for the design of HIV-1 inhibitors. We employed a comprehensive set of computational tools, predominantly based on free energy calculations and molecular-dynamics simulations, to investigate the molecular recognition of CXCR4 by a dual tropic V3 loop. We report what is, to our knowledge, the first HIV-1 gp120 V3 loop:CXCR4 complex structure. The computationally derived structure reveals an abundance of polar and nonpolar intermolecular interactions contributing to the HIV-1 gp120:CXCR4 binding. Our results are in remarkable agreement with previous experimental findings. Therefore, this work sheds light on the functional role of HIV-1 gp120 V3 loop and CXCR4 residues associated with HIV-1 coreceptor activity.     

Distinct mechanisms of CD4+ and CD8+ T-cell activation and bystander apoptosis induced by human immunodeficiency virus type 1 virions

Apoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4(+) and CD8(+) T cells. Infection of primary T-cell cultures with ELI6 induced CD4(+) T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4(+) and CD8(+) T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4(+) T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8(+) T cells was triggered by a soluble factor(s) secreted by CD4(+) T cells. HIV-1 virions activated CD4(+) and CD8(+) T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25(+)HLA-DR(+) T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4(+) and CD8(+) T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.     

Segregation of CD4 and CXCR4 into Distinct Lipid Microdomains in T Lymphocytes Suggests a Mechanism for Membrane Destabilization by Human Immunodeficiency Virus

Recent evidence has suggested that plasma membrane sphingolipids and cholesterol spontaneously coalesce into raft-like microdomains and that specific proteins, including CD4 and some other T-cell signaling molecules, sequester into these rafts. In agreement with these results, we found that CD4 and the associated Lck tyrosine kinase of peripheral blood mononuclear cells and H9 leukemic T cells were selectively and highly enriched in a low-density lipid fraction that was resistant at 0 degrees C to the neutral detergent Triton X-100 but was disrupted by extraction of cholesterol with filipin or methyl-beta-cyclodextrin. In contrast, the CXCR4 chemokine receptor, a coreceptor for X4 strains of human immunodeficiency virus type 1 (HIV-1), was almost completely excluded from the detergent-resistant raft fraction. Accordingly, as determined by immunofluorescence with confocal microscopy, CD4 and CXCR4 did not coaggregate into antibody-induced cell surface patches or into patches of CXCR4 that formed naturally at the ruffled edges of adherent cells. The CXCR4 fluorescent patches were extracted with cold 1% Triton X-100, whereas the CD4 patches were resistant. In stringent support of these data, CD4 colocalized with patches of cholera toxin bound to the raft-associated sphingoglycolipid GM1, whereas CXCR4 did not. Addition of the CXCR4-activating chemokine SDF-1 alpha did not induce CXCR4 movement into rafts. Moreover, binding of purified monomeric gp120 envelope glycoproteins from strains of HIV-1 that use this coreceptor did not stimulate detectable redistributions of CD4 or CXCR4 between their separate membrane domains. However, adsorption of multivalent gp120-containing HIV-1 virion particles appeared to destabilize the local CD4-containing rafts. Indeed, adsorbed HIV-1 virions were detected by immunofluorescence microscopy and were almost all situated in nonraft regions of the cell surface. We conclude that HIV-1 initially binds to CD4 in a raft domain and that its secondary associations with CXCR4 require shifts of proteins and associated lipids away from their preferred lipid microenvironments. Our evidence suggests that these changes in protein-lipid interactions destabilize the plasma membrane microenvironment underlying the virus by at least several kilocalories per mole, and we propose that this makes an important contribution to fusion of the viral and cellular membranes during infection. Thus, binding of HIV-1 may be favored by the presence of CD4 in rafts, but the rafts may then disperse prior to the membrane fusion reaction.     

Ligand binding characteristics of CXCR4 incorporated into paramagnetic proteoliposomes

The G protein-coupled receptor CXCR4 is a coreceptor, along with CD4, for the human immunodeficiency virus type 1 (HIV-1) and has been implicated in breast cancer metastasis. We studied the binding of the HIV-1 gp120 envelope glycoprotein (gp) to CXCR4 but found that the gp120s from CXCR4-using HIV-1 strains bound nonspecifically to several cell lines lacking human CXCR4 expression. Therefore, we constructed paramagnetic proteoliposomes (CXCR4-PMPLs) containing pure, native CXCR4. CXCR4-PMPLs specifically bound the natural ligand, SDF-1alpha, and the gp120s from CXCR4-using HIV-1 strains. Conformation-dependent anti-CXCR4 antibodies and the CXCR4 antagonist AMD3100 blocked HIV-1 gp120 binding to CXCR4-PMPLs. The gp120-CXCR4 interaction was blocked by anti-gp120 antibodies directed against the third variable (V3) loop and CD4-induced epitopes, structures that have also been implicated in the binding of gp120 to the other HIV-1 coreceptor, CCR5. Compared with the binding of R5 HIV-1 gp120s to CCR5, the gp120-CXCR4 interaction exhibited a lower affinity (K(d) = 200 nm) and was dependent upon prior CD4 binding, even at low temperature. Thus, although similar regions of X4 and R5 HIV-1 gp120s appear to be involved in binding CXCR4 and CCR5, respectively, differences exist in nonspecific binding to cell surfaces, affinity for the chemokine receptor, and CD4 dependence at low temperature.     

Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection

HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4⁺ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1.     

Evidence for Common Structural Determinants of Human Immunodeficiency Virus Type 1 Coreceptor Activity Provided through Functional Analysis of CCR5/CXCR4 Chimeric Coreceptors

Human immunodeficiency virus type 1 (HIV-1) infection in vivo is dependent upon the interaction of the viral envelope glycoprotein gp120 with CC chemokine receptor 5 (CCR5) or CXC chemokine receptor 4 (CXCR4). To study the determinants of the gp120-coreceptor association, we generated a set of chimeric HIV-1 coreceptors which express all possible combinations of the four extracellular domains of CCR5 and CXCR4. Stable U87 astroglioma cell lines expressing CD4 and individual chimeric coreceptor proteins were tested against a variety of R5, X4, and R5X4 envelope glycoproteins and virus strains for their ability to support HIV-1-mediated cell fusion and infection, respectively. Each of the cell lines promoted fusion with cells expressing an HIV envelope glycoprotein, except for U87.CD4.5455, which presents the first extracellular loop (ECL1) and flanking sequences of CXCR4 in the context of CCR5. However, all of the chimeric coreceptors allowed productive infection by one or more of the viral strains tested. Viral phenotype was a predictive factor for the observed activity of the chimeric molecules; X4 and R5X4 HIV strains utilized a majority of the chimeras, while R5 strains were limited in their ability to infect cells expressing these chimeric molecules. The expression of CCR5 ECL2 within the CXCR4 backbone supported infection by an R5 primary isolate, but no chimeras bearing the N terminus of CCR5 exhibited activity with R5 strains. Remarkably, the introduction of any CXCR4 domain into the CCR5 backbone was sufficient to allow utilization by multiple X4 strains. However, critical determinants within ECL2 and/or ECL3 of CXCR4 were apparent for all X4 viruses upon replacement of these domains in CXCR4 with CCR5 sequences. Unexpectedly, chimeric coreceptor-facilitated entry was blocked in all cases by the presence of the CXCR4-specific inhibitor AMD3100. Our data provide proof that CCR5 contains elements that support usage by X4 viral strains and demonstrate that the gp120 interaction sites of CCR5 and CXCR4 are structurally related.     

Filamin-A regulates actin-dependent clustering of HIV receptors

Human immunodeficiency virus (HIV)-1 infection requires envelope (Env) glycoprotein gp120-induced clustering of CD4 and coreceptors (CCR5 or CXCR4) on the cell surface; this enables Env gp41 activation and formation of a complex that mediates fusion between Env-containing and target-cell membranes. Kinetic studies show that viral receptors are actively transported to the Env-receptor interface in a process that depends on plasma membrane composition and the actin cytoskeleton. The mechanisms by which HIV-1 induces F-actin rearrangement in the target cell remain largely unknown. Here, we show that CD4 and the coreceptors interact with the actin-binding protein filamin-A, whose binding to HIV-1 receptors regulates their clustering on the cell surface. We found that gp120 binding to cell receptors induces transient cofilin-phosphorylation inactivation through a RhoA-ROCK-dependent mechanism. Blockade of filamin-A interaction with CD4 and/or coreceptors inhibits gp120-induced RhoA activation and cofilin inactivation. Our results thus identify filamin-A as an adaptor protein that links HIV-1 receptors to the actin cytoskeleton remodelling machinery, which may facilitate virus infection.     

Matrix fibronectin increases HIV stability and infectivity

HIV particles are detected extracellularly in lymphoid tissues, a major reservoir of the virus. We previously reported that a polymerized form of fibronectin (FN), superfibronectin (sFN), as well as a fragment of FN, III1-C, enhanced infection of primary CD4(+) T cells by HIV-1IIIB. We now show that sFN enhances infection of primary CD4(+) T cells by both R5 and X4 strains of HIV-1. Using HIV pseudotyped with different envelope glycoproteins (gp120) and HOS cells transfected with various chemokine receptors alone or in combination with the CD4 molecule, we show that sFN-mediated enhancement requires the CD4 receptor and does not alter the specificity of gp120 for different chemokine receptors. Because the III1-C fragment also resulted in enhancement, we asked whether proteolysis of FN generated fragments capable of enhancing HIV infection. We found that progressive proteolysis of FN by chymotrypsin correlates with an enhancement of HIV infection in both primary CD4(+) T cells and the IG5 reporter cell line. Furthermore, incubation of HIV with sFN significantly prolonged infectivity at 37 degrees C compared with dimeric FN or BSA. In conclusion, these results indicate that polymerized (matrix) or degraded (inflammation-associated), but not dimeric (plasma), FN are capable of enhancing infection by HIV-1, independent of the coreceptor specificity of the strains. Moreover, virions bound to matrix FN maintain infectivity for longer periods of time than do virions in suspension. This study suggests that matrix proteins and their conformational status may play a role in the pathogenesis of HIV.